Iron transport protein

ABSTRACT

This invention is directed to a novel protein of general significance to the cellular transport of nontransferrin bound iron. This heretofore unidentified protein, which is termed &#34;stimulator of Fe (iron) transport&#34; (SFT) protein, is essential to an alternative pathway of iron uptake. The cDNA encoding the SFT protein has been cloned and sequenced. The gene for the SFT protein has been mapped to chromosome 10, 17% of the distance from the centromere to the telomere of chromosome arm 10q, an area that corresponds to band 10q21. In addition, the amino acid structural identity of the SFT protein has been deduced, represented in a predicted transmembrane structure with a 6-transmembrane motif.

This application is a continuation of provisional application No.60/019,528, filed Jun. 6, 1996 now abandoned, and a continuation ofprovisional application No. 60/056,915, filed Feb. 26, 1996 nowabandoned.

This invention was supported by NIH Grant Nos. DK45737 and DK07703, andthe government has certain rights to the invention.

This application is a continuation of provisional application No.60/019,528, filed Jun. 6, 1996 now abandoned, and a continuation ofprovisional application No. 60/056,915, filed Feb. 26, 1996 nowabandoned.

This invention was supported by NIH Grant Nos. DK45737 and DK07703, andthe government has certain rights to the invention.

FIELD OF THE INVENTION

This invention is in the field of cellular physiology, and inparticular, relates to iron transport in cells. The novel protein andcompositions of the invention are useful in modulating iron uptake incells.

BACKGROUND OF THE INVENTION

Ionic iron is an essential metal for the growth and maintenance ofanimals and most microorganisms. In humans, iron is required for oxygenmetabolism, hemoglobin production, and electron transfer reactions,among many other reactions. Ionic iron is actively transported in to themucosal cells of the intestine, where it binds to the protein ferritin.This phenomenon is called the mucosal iron barrier. The iron-ferritincomplex then serves as a local intracellular storehouse for iron. Whenbody reserves of iron are adequate, very little iron is allowed to passinto the portal blood, and most of the stored iron is lost as theepithelial cells later slough off. As iron reserves are depleted, asoccurs during acute or chronic hemorrhage, iron uptake from theintestine and its release to the blood are accelerated.

In blood, iron binds to transferrin, a plasma protein that transports itto cells. Transferrin bound iron is delivered to cells, by binding totransferrin cell surface receptors and is endocytosed into acidicintracellular compartments. The low pH of intracellular endosomaldomains promotes the release of iron from transferrin while bound to itsreceptor. Recent studies indicate, however, that there is an alternativepathway for cellular iron transport, which is independent of thetransferrin mediated pathway. In this pathway, non-transferrin boundiron uptake does not appear to be influenced by intracellular ironlevels. Inman and Wessling-Resnick, I. Biol. Chem., 268:8521-8528(1993).

Iron deficiencies can cause iron deficient anemia in a patient. Irondeficiency also results in gastrointestinal problems, anorexia, andparesthesia. Anemia is usually treated with a combination therapy ofdiet, iron supplements, and additional vitamins, such as vitamin B-12and folic acid to increase the absorption of iron. Pregnant women inparticular are most often provided with iron supplements to guardagainst iron-deficiency.

There are several disorders that are the result of iron overload,including damage to the liver (cirrhosis), the heart, and the pancreas.Hemochromatosis, a genetic disorder with approximately one in twentypeople carrying the mutant gene for genetic hemochromatosis, results inexcess iron being deposited in the tissues and in increased incidence ofhepatic cancer and liver cirrhosis, although clinical symptoms ofpatients homozygous for this disease may vary. Another hereditarydisease characterized by chronic iron overload is Cooley's anemia(Thallasemia major), where congestive heart failure often precedes rapiddeterioration and death of the untreated patient almost always in earlyinfancy. In addition, repeated blood transfusions may causetransfusional siderosis, the accumulation of excess iron in the body.The iron liberated from the transfused cells cannot be excreted andaccumulates in the cells of the reticuloendothelial system and incardiac muscle, kidneys, thyroid gland and adrenal gland. Changes iniron distribution from the primary reticuloendothelial iron toparenchymal iron overload are ascribed to the high saturation oftransferrin, which provides favorable conditions for uptake of iron byparenchymal cells. Free transferrin thus protects the tissues fromsiderosis.

Iron (III) chelators are used in the treatment of iron overload and ofinfectious diseases caused by iron-dependent pathogens. Iron chelatorcompounds work by forming a stable complex that prevents the iron fromentering into further chemical reactions. Different iron chelators havedifferent modes of action. Deferoxamine mesylate chelates iron fromferritin and hemosiderin, but not readily from transferring it does notcombine with the iron from cytochromes and hemoglobin. A disadvantage ofdeferoxamine mesylate is that it can render the patient more susceptibleto infections from bacteria and fungi, which can obtain iron directlyfrom their environment or by secretion of low molecular weightsiderophores that bind iron (III) at high affinity and return to thecell surface where iron delivery occurs via receptor mediated uptake.

It is a continuing goal of researchers to develop pharmaceutics whichcan regulate the levels of cellular iron and thus treat iron deficientconditions and iron overload disorders and diseases.

SUMMARY OF THE INVENTION

This invention is directed to a novel protein of general significance tothe cellular transport of nontransferrin bound iron. This heretoforeunidentified protein, which is termed "stimulator of Fe (iron)transport" (SFT) protein, is essential to an alternative pathway of ironuptake. The cDNA encoding the SFT protein has been cloned and sequenced.The gene for the SFT protein has been mapped to chromosome 10, 17% ofthe distance from the centromere to the telomere of chromosome arm 10q,an area that corresponds to band 10q21. In addition, the amino acidstructural identity of the SFT protein has been deduced, represented ina predicted transmembrane structure with a 6-transmembrane motif.

In one aspect, then, the invention is directed to purified and isolatedpeptide domains comprising the SFT protein and to molecules that mimicits structure and/or function, which may be used for inducing ormodulating iron uptake. Chemical compounds that disrupt the function ofthe SFT protein have utility as iron uptake modulating agents.Accordingly, in another aspect, the invention is direct to agentscapable of disrupting SFT protein function. These agents include, butare not limited to, molecules that bind to the SFT protein, moleculesthat interfere with the interaction of the SFT protein with otherprotein(s), and molecules comprising the SFT protein which is altered insome manner. The invention provides methods to identify molecules thatmodulate iron uptake by disrupting the function of the SFT protein,which accordingly comprise additional contemplated embodiments.

In additional aspects, the present invention relates to products andprocesses involved in the cloning, preparation and expression of peptidedomains comprising the SFT protein; antibodies with specificity to theSFT protein; and nucleotide sequences encoding the SFT protein orportions thereof. Peptide domains comprising the SFT protein are usefulfor producing antibodies thereto. These antibodies are useful fordetecting and isolating proteins comprising the SFT protein inbiological specimens, including for example, cells from all humantissue, especially the liver, the spleen, the intestines, and bonemarrow.

In yet another aspect, the invention provides for expression vectorscontaining genetic sequences, hosts transformed with such expressionvectors, and methods for producing the recombinant SFT protein of theinvention.

The invention relates to the therapeutic use of peptides comprising theSFT protein.

The invention also relates to methods for modulating the iron uptake ofa patient by administering peptides comprising the SFT protein, oranalogues thereof, to a patient suffering from deficient iron uptake inorder to restore normal iron uptake.

The structural information of the SFT protein may be used to developinterventions against iron deficient anemias and iron overload. Thus,pharmaceuticals may be devised based on the SFT structure to eitherstimulate or block iron uptake.

These and other objects and aspects of the invention will be apparent tothose of skill from the description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of the SFT protein. SEQ ID NO. 1.

FIG. 2 is the amino acid sequence of the SFT protein. SEQ ID NO. 2.

DETAILED DESCRIPTION OF THE INVENTION

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of skill in the art publicationsand other materials setting forth such known methodologies to whichreference is made are incorporated herein by reference in theirentireties as though set forth in full. Standard reference works settingforth the general principles of recombinant DNA technology includeSambrook, J., et al., Molecular Cloning: A Laboratory Manual 2d Ed.,Cold Spring Harbor Laboratory press, Planview, N.Y. (1989); Mcpherson,M. J., Ed., Directed Mutagenesis: A practical Approach, IRL press,Oxford (1991); Jones, J., Amino Acid and peptide Synthesis, OxfordScience publications, Oxford (1992); Austen, B. M. and Westwood, O. M.R., Protein Targeting and Secretion, IRL press, Oxford (1991). Anysuitable materials and/or methods known to those of skill can beutilized in carrying out the present invention; however, preferredmaterials and/or methods are described. Materials, reagents and the liketo which reference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

A previously unidentified protein that appears to be both necessary andsufficient for nontransferrin bound iron uptake has now been identified.This protein, designated herein as the "SFT protein," (stimulator of Fe(iron) transport) mediates iron uptake function. As those of skillfamiliar with the present invention will appreciate, peptide domainscomprising the SFT protein are useful in modulating iron uptake incells. Similarly, compounds and compositions which are capable ofbinding to the SFT protein are useful as agents for the modulation ofiron uptake activity in cells.

As used herein, the term "SFT protein " refers to a protein domain firstidentified in a human erythroleukemia cell line (K562), demonstratedherein to be essential for iron uptake; and to peptide domains and/ormolecules capable of mimicking its structure and/or function. In apreferred embodiment, the present invention comprises a protein havingthe amino acid sequence identified in SEQ ID NO. 2, as well asfunctional equivalents thereof. By "functional equivalent" is meant apeptide domain possessing a biological activity or immunologicalcharacteristic substantially similar to that of the SFT protein, and isintended to include "fragments", "variants", "analogs", "homologs", or"chemical derivatives" possessing such activity or characteristic.Functional equivalents of the SFT domains, then, may not share anidentical amino acid sequence, and conservative or non-conservativeamino acid substitutions of conventional or unconventional amino acidsare possible.

Reference herein to "conservative" amino acid substitution is intendedto mean the interchangeability of amino acid residues having similarside chains. For example, glycine, alanine, valine, leucine andisoleucine make up a group of amino acids having aliphatic side chains;serine and threonine are amino acids having aliphatic-hydroxyl sidechains; asparagine and glutamine are amino acids having amide-containingside chains; phenylalanine, tyrosine and tryptophan are amino acidshaving aromatic side chains; lysine, arginine and histidine are aminoacids having basic side chains; and cysteine and methionine are aminoacids having sulfur-containing side chains. Interchanging one amino acidfrom a given group with another amino acid from that same group would beconsidered a conservative substitution. Preferred conservativesubstitution groups include asparagine-glutamine, alanine-valine,lysine-arginine, phenylalanine-tyrosine and valine-leucine-isoleucine.

Agents capable of modulating iron transport or iron uptake mediated bythe SFT protein may include peptide domains comprising the SFT protein,as well as mutants of the SFT protein. A "mutant" as used herein refersto a peptide having an amino acid sequence which differs from that ofthe naturally occurring peptide or protein by at least one amino acid.Mutants may have the same biological and immunological activity as thenaturally occurring SFT protein. However, the biological orimmunological activity of mutants may differ or be lacking. For example,a SFT protein mutant may lack the biological activity whichcharacterizes naturally occurring SFT protein, but may be useful as anantigen for raising antibodies against the SFT protein or for thedetection or purification of antibodies against the SFT protein, or asan agonist (competitive or non-competitive), antagonist, or partialagonist of the function of the naturally occurring SFT protein.

Modulation of iron uptake or iron transport mediated by the SFT proteinmay be effected by agonists or antagonists of SFT protein as well.Screening of peptide libraries, compound libraries and other informationgene banks to identify agonists or antagonists of the function ofproteins comprising the SFT protein is accomplished with assays fordetecting the ability of potential agonists or antagonists to inhibit oraugment SFT iron transport.

For example, high through-put screening assays may be used to identifycompounds that modulate the iron transport function of the SFT protein.These screening assays facilitate the identification of compounds thataccelerate or inhibit iron transport by influencing iron transportmediated by the SFT protein. For example, an in vitro screen forcompounds that disrupt the SFT protein interaction with iron comprisesmultiwell plates coated with SFT protein which are incubated with alabeled iron in the presence of one or more compounds to be tested.Molecules that specifically disrupt the interaction could, in principle,bind to either the SFT protein "ligand" or interfere with the labellediron. Either class of compound would be a candidate iron modulatingagent.

A high speed screen for agents that bind directly to the SFT protein mayemploy immobilized or "tagged" combinatorial libraries. Agents that bindspecifically to such libraries are candidates to be tested for theircapacity to block iron uptake activity. As discussed above, such agentsmay function as suppressors of iron uptake by directly inhibiting SFTprotein function. These agents would be useful for suppressing aberrantiron uptake in iron overload disorders and diseases.

Antibodies against the SFT protein of the invention may be used toscreen cDNA expression libraries for identifying clones containing cDNAinserts encoding structurally related, immunocrossreactive proteinswhich may be members of the family of SFT protein. Screening of cDNA andmRNA expression libraries is known in the art. Similarly, antibodiesagainst the SFT protein are used to identify or purifyimmunocrossreactive proteins related to this peptide, or to detect ordetermine the amount of proteins containing the SFT protein in a cell orcell population, for example, in tissue or cells, such as hepatocytes.Known methods for such measurements include immunoprecipitation of cellextracts followed by PAGE, in situ detection by immunohistochemicalmethods, and ELISA methods, all of which are well known in the art.

Modulation of iron uptake according to the invention includes methodsemploying specific antisense polynucleotides complimentary to all orpart of the nucleotide sequences encoding peptide domains comprising theSFT protein or antisense polynucleotides complimentary to all or part ofthe 3' or 5' noncoding regions of the SFT protein. Such complimentaryantisense polynucleotides may include nucleotide additions, deletions,substitutions and transpositions, providing that specific hybridizationto the target sequence persists. Soluble antisense RNA or DNAoligonucleotides which can hybridize specifically to mRNA speciesencoding proteins comprising the SFT protein, and which preventtranscription of the mRNA species and/or translation of the encodedpolypeptide are contemplated as complimentary antisense polynucleotidesaccording to the invention. Production of peptide domains comprising theSFT protein is inhibited by antisense polynucleotides according to theinvention, and such antisense polynucleotides may inhibit iron uptake,and/or reverse the transformed phenotype of cells. In addition,riboregulators which effectively prohibit the transcription ortranslation of a protein may also be employed as an antisensepolynucleotide. A heterologous expression cassette maybe used to produceantisense polynucleotides in a transfectant or transgenic cell.Antisense polynudeotides also may be administered as solubleoligonucleotides to the external environment of the target cell, such asthe culture medium of cells in vitro or the interstitial fluid (e.g.,via the circulatory system) in vivo. Antisense polynucleotides and theiruse are known to those of skill, and are described, for example, inMelton, D. A., Ed, Antisense RNA and DNA, Cold Spring Harbor Laboratorypress, Cold Spring Harbor, N.Y. (1988).

Computer-aided molecular modeling of the SFT protein can be used tostudy its three-dimensional structures using computer visualizationtechniques. Novel designs of low molecular weight inhibitors oroligopeptides can then be analyzed for selective inhibition.Descriptions of targeted drug design can be found in I. Kuntz,"Structure-Based Strategies for Drug Design and Discovery," Science,257:1078-1082 (1992) and J. Dixon, "Computer-Aided Drug Design: Gettingthe Best Results," Trends in Biotechnology 10:357-363 (1992). Specificapplications of the binding of inhibitors to targets using computermodeling have been described in piper et al., "Studies Aided byMolecular Graphics of Effects of Structural Modifications on the Bindingof Antifolate Inhibitors to Human Dihydrofolate Reductase," Proc Am.Assoc. Cancer Res. Annual Meeting, 33:412 (1992); Hibert et al.,"Receptor 3D-Models and Drug Design," Therapie (Paris), 46:445-451(1991)(serotonin receptor recognition sites). Computer programs that canbe used to conduct three-dimensional molecular modeling are described inG. Klopman, "Multicase 1: A Hierarchical Computer Automated StructureEvaluation Program," Quantitative Structure-Activity Relationships,11:176-184 (1992); pastor et al., "The Edisdar programs Rational DrugSeries Design," Quantitative Structure-Activity Relationships,10:350-358 (1991); Bolis et al., "A Machine Learning Approach toComputer-Aided Molecular Design," J . Computer Aided Molecular Design,5:617-628 (1991); and Lawrence and Davis, "CLIX: A Search Algorithm forFinding Novel Ligands Capable of Binding proteins of KnownThree-Dimensional Structure," Proteins Structure Functional Genetics12:31-41 (1992).

Low molecular weight inhibitors specific for the SFT protein can bepredicted by molecular modeling and synthesized by standard organicchemistry techniques. Computer modeling can identify oligopeptides whichblock the iron transport activity. Techniques for producing theidentified oligopeptides are well known and can proceed by organicsynthesis of amino acids, by genetic engineering techniques, or by PCRbased amplification. R. Silverman, The Organic Chemistry of Drug Designand Drug Action, Academic Press (1992).

The inhibitors of this invention can be identified by selecting thoseinhibitors that selectively inhibit the iron transport ability of theSFT protein.

Peptidomimetics of the SFT protein are also provided by the presentinvention, and can act as drugs for the modulation of iron uptake by,for example, blocking or interfering with the function of peptidedomains comprising the SFT protein. peptidomimetics are commonlyunderstood in the pharmaceutical industry to include non-peptide drugshaving properties analogous to those of those of the mimicked peptide.The principles and practices of peptidomimetic design are known in theart and are described, for example, in Fauchere J., Adv. Drug Res. 15:29(1986); and Evans et al., J. Med. Chem. 30:1229 (1987). Peptidomimeticswhich bear structural similarity to therapeutically useful peptides maybe used to produce an equivalent therapeutic or prophylactic effect.Typically, such peptidomimetics have one or more peptide linkagesoptionally replaced by a linkage which may convert desirable propertiessuch as resistance to chemical breakdown in vivo. These linkages mayinclude --CH₂ NH--, --CH₂ S--, --CH₂ --CH₂ --, --CH=CH--, --COCH₂ --,--CH(OH)CH₂ --, and --CH₂ SO--. Peptidomimetics may exhibit enhancedpharmacological properties (biological half life, absorption rates,etc.), different specificity, increased stability, production economies,lessened antigenicity and the like which makes their use as therapeuticsparticularly desirable.

It is possible, then, to employ the invention for detection ordetermination of peptide domains comprising the SF1 protein, forexample, in fractions from tissue/organ excisions, by means ofimmunochemical or other techniques in view of the antigenic propertiesthereof. Immunization of animals with peptide domains comprising the SFTprotein alone or in conjunction with adjuvants by known methods canproduce antibodies specific for the SFT protein. Antiserum obtained byconventional procedures may be utilized for this purpose. For example, amammal, such as a rabbit, may be immunized with a peptide domaincomprising the SFT protein, thereby inducing the formation of polyclonalantibodies thereagainst. Monoclonal antibodies also may be generatedusing known procedures. Such antibodies can be used according to theinvention to detect the presence and amount of peptide domainscomprising the SFT protein.

The SFT protein and other compositions of the present invention may beproduced by recombinant DNA techniques known in the art. For example,nucleotide sequences encoding the SFT protein of the invention may beinserted into a suitable DNA vector, such as a plasmid, and the vectorused to transform a suitable host. The recombinant SFT protein isproduced in the host by expression. The transformed host may be aprokaryotic or eukaryotic cell. Preferred nucleotide sequences for thispurpose encoding the SFT protein is set forth in SEQ ID NO. 1.

polynucleotides encoding peptides comprising the SFT protein may begenomic or cDNA, isolated from clone libraries by conventional methodsincluding hybridization screening methods. Alternatively, syntheticpolynucleotide sequences may be constructed by known chemical syntheticmethods for the synthesis of oligonucleotides. These synthetic methodsare described, for example, in Blackburn, G. M. and Gait, M. J., Ed.,Nucleic Acids in Chemistry and Biology, IRL press, Oxford, England(1990), and it will be evident that commercially availableoligonudeotide synthesizers also may be used according to themanufacturer's instructions.

polymerase chain reaction (PCR) using primers based on the nucleotidesequence data disclosed herein may be used to amplify DNA fragments frommRNA pools, cDNA clone libraries or genomic DNA. PCR nucleotideamplification methods are known in the art and are described, forexample, in Erlich, H. A., Ed., PCR Technology: Principles andApplications for DNA Amplification, Stockton press, New York, N.Y.(1989); U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159; and U.S. Pat.No. 4,683,195. Various nucleotide deletions, additions and substitutionsmay be incorporated into the polynucleotides of the invention as will berecognized by those of skill, who will also recognize that variation inthe nucleotide sequence encoding SFT protein may occur as a result of,for example, allelic polymorphisms, minor sequencing errors, and thelike. The polynucleotides encoding SFT protein of the invention mayinclude short oligonucleotides which are useful, for example, ashybridization probes and PCR primers. The polynucleotide sequences ofthe invention also may comprise a portion of a larger polynucleotideand, through polynucleotide linkage, they may be fused, in frame, withone or more polynucleotide sequences encoding different proteins. Inthis event, the expressed protein may comprise a fusion protein. Ofcourse, the polynucleotide sequences of the invention may be used in thePCR method to detect the presence of mRNA encoding SFT protein in thediagnosis of disease or in forensic analysis.

cDNAs encoding proteins which interact with the SFT protein (or proteinscontaining the SFT protein) can be identified by screening cDNAexpression libraries, employing known methods. Examples of such methodsinclude the yeast two-hybrid system (Chien, et al., proc. Nat'l. Acad.Sci. 88: 9578 (1991), and the E. coli/BCCp interactive screening system(Germino, et al., proc. Nat'l. Acad. Sci. 90: 1639 (1993). Suitable cDNAlibraries will include mammalian cDNA libraries, such as human, mouse orrat, which may contain cDNA produced from RNA and a single cell, tissueor organ type or developmental stage, as are known in the art.

A nucleotide sequence encoding the SFT protein or a peptide domainthereof may be inserted into a DNA vector in accordance withconventional techniques, including blunt-ending or staggered-endingtermini for ligation, restriction enzyme digestion to provideappropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed, for example, by Sambrook, et al., (In: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y. Second Edition (1989)), and are well known in the art.

The rational design of SFT protein mimetics or binding molecules, basedon modeled (or experimentally determined) peptide structure, may becarried out by those of skill, using known methods of rational drugdesign. Therapeutic or prophylactic methods for treating iron overloadand the like, are accomplished by the administration of an effectiveamount of a therapeutic agent capable of specifically inhibiting SFTprotein, thereby modulating the iron transport activity of the SFTprotein.

Any mode of administration which results in the delivery of thetherapeutic agent across the cell membrane and into the desired cell iscontemplated as within the scope of the present invention. The site ofadministration and cells will be selected by one of ordinary skill inthe art based upon an understanding of the particular degenerativedisorder being treated. In addition, the dosage amount, dosagefrequency, and length of course of treatment, can be determined andoptimized by one of ordinary skill in the art depending upon theparticular disorder being treated. The particular mode of administrationcan also be readily selected by one of ordinary skill in the art and caninclude, for example, oral, intravenous, subcutaneous, intramuscular,etc., with the requirement that the therapeutic agent cross the cellmembrane. Principles of pharmaceutical dosage and drug delivery areknown and are described, for example, in Ansel, H. C. and Popovich, N.G., Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition,Lea & Febiger, Publisher, Philadelphia, Pa. (1990). It is possible, forexample, to utilize liposomes to specifically deliver the agents of theinvention. Liposomes can be produced so that they contain additionalbioactive compounds and the like, such as drugs, radioisotopes, lectinsand toxins, which would act at the target site.

Suitable agents for use according to the invention include SFT proteinand mimetics, peptide domains, fragments, functional equivalents and/orhybrids or mutants thereof, as well as vectors containing cDNA encodingany of the foregoing. Agents can be administered alone or in combinationwith and/or concurrently with other suitable drugs and/or courses oftherapy.

In addition, cells and non-human transgenic animals having one or morefunctionally impaired alleles encoding the SFT protein or peptide domainmay be generated using homologous targeting constructs from genomicclones. Methods for the production of homologous targeting constructsare known and described, for example, in Bradley, et al., Bio/Technology10:534 (1992); and Koh, et al., Science 256: 1210 (1992). For example,"knock-out" mice may be generated which are homozygous or heterozygousfor an inactivated allele of a protein comprising the SFI protein by useof homologous targeting. These mice are useful as research subjects forthe investigation of disease and for other uses. Methods of producingchimeric targeted mice are known and are described, for example, inRobertson, E. J., Ed., Teratocarcinomas and Embryonic Stem Cells: Apractical Approach, IRL Press, Washington, D.C. (1987), which alsodescribes the manipulation of embryonic stem cells. In addition,transgenes for expressing polypeptides comprising the SFT protein athigh levels or under the control of selected transcription controlsequences may be constructed using the cDNA or genomic gene of a proteincomprising the SFF protein. Transgenes so constructed can be introducedinto cells and transgenic non-human animals by known methods. Suchtransgenic cells and transgenic non-human animals may be used as screensfor agents which modulate iron uptake.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention in any way.

EXAMPLES

The present inventors have derived from molecular cloning of the humanstimulator of Fe (iron) transport (SFT) cDNA the complete primarystructure of human SFT. SFT is ubiquitously expressed indicating thatthe activity of this protein is most likely an essential element of irontransport in all tissues. Since iron is a required essential nutrientfor cell survival and growth, SFT appears to play a key role in ironmetabolism and is likely to be regulated in states of iron-deficientanemia and iron overload (hemochromatosis).

Isolation of RNA and Synthesis of First Strand cDNA: Total RNA wasisolated from human K562 erythroleukemia cells stimulated by phorbolesters to enhance iron uptake (Akompong et al., J. Biol. Chem., 270:20937-20941 (1995)) using the guanidinium isothiocyanate method(Chirgwin et al., Biochemistry, 18: 5294-5299). polyadenylated RNA wasisolated using the Invitrogen "FastTrack" System (Invitrogen, San Diego,Calif.). First-strand cDNA synthesis from the human K562 cellpolyadenylated RNA was accomplished using an oligo-dT₁₇ primercontaining a NotI restriction at the 5' end (Promega, Madison, Wis.)using procedure described by Gubler and Hofman, Gene, 25: 263.

Functional Expression Cloning of SFT cDNA: Human K562 cell cDNA wasdirectionally subcloned into pBluecript SK(-) (Stratagene, La Jolla,Calif.). Briefly, EcoRI linkers were ligated to the 5' ends of the cDNAwhich was subsequently digested with NotI. This K562 cell cDNA librarywas transformed into XLIBlue cells (Stratagene, La Jolla, Calif.).Approximately forty-thousand independent colonies of these cells wereseparated into 5 pools for plasmid isolation. The plasmid cDNAs werethen subject to in vitro transcription and capping using the "mCap" kit(Stratagene, La Jolla, Calif.). Transcripts (˜20 to 25 ng) prepared fromthese pools were then injected both individually in combination intooocytes isolated from Xenopus laevis. After a 48 hour incubation at 20°C. to permit expression of proteins from the injected cRNAs, oocyteswere subsequently assayed for the ability to take up radioactive ironusing methods adapted from Inman and Wessling-Resnick, J. Biol. Chem,268: 8521-8528 (1993). XLIBlue cells identified to carry plasmid whichdirected transcription of the apparent iron transport activity werefurther subdivided into a second set of 5 pools for plasmid isolationand preparation of cRNAs to inject into Xenopus oocytes for irontransport assays. Iterative screening in this manner led to theisolation of a single 1403 base pair clone with which iron transportactivity could be expressed when co-injected with a second pool of fourindependent clones. Sequencing of both strands of the 1403 base pairclone was performed with flanking and internal primers by the dideoxychain-termination method of Sanger et al., proc. Nat'l. Acad. Sci. USA,74: 5463-5467.

Sequence data was analyzed using programs available through the NationalInstitutes of Health sponsored Molecular Biology Computer ResearchResource of the Dana Farber Cancer Institute/Harvard School of publicHealth. A search using the BLAST algorithm (Altschul et al. J. Mol.Biol., 215: 403) of all available data bases (including non-redundantPDB, GBupdate, GenBank, EMBLupdate, and EMBL) revealed that this clonewas a heretofore unidentified species. A single open reading framepredicted to encode a 338 amino acid protein was identified and furthersubcloned into the transcription-computer vector pAGA (Sanford et al.,J. Biol. Chem., 266: 9570-9579). Following in vitro transcription andcapping, cRNAs containing the open reading frame were injected intoXenopus oocytes. Iron transport assays demonstrated that injection ofthis transcript alone (absent of 5' and 3' regions of the originalfull-length clone) was sufficient to confer iron uptake activity to theoocytes, thus confirming-that the identified coding sequence dictatedsynthesis for SFT itself.

Northern Analysis: A commercially available multiple tissue Northernblot containing various human tissues (Clontech, Palo Alto, Calif.) wasused for analysis of SFT expression with methods described by themanufacturer. As a probe, the 1403 bp fragment of the SFT gene waslabelled with 32P!-dCTP by random priming (Pharmacia Oligolabelling Kit,Pharmacia, Piscataway, N.J.). Two mRNAS of ˜2.4 and ˜1.5 kilobases werehybridized by the SFT probe and were present in all tissues examinedincluding spleen, thymus, intestine, and peripheral blood leukocytes.

Miscellaneous: All molecular biology protocols employed, includingrestriction enzyme digests, ligation reactions, DNA sequencing, plasmidisolation, etc., were standard techniques described, for example, inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press, Planview, N. Y. (1989).

Deposit Information: A culture containing the SFT cDNA was depositedwith the American Type Culture Collection (ATCC) at 12301 ParklawnDrive, Rockville, Md. 20852 USA on Mar. 20, 1996.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious to one skilled in the art that certainchanges and modifications may be practiced within the scope of theappended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1395 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCGGCTGTCGCACTTACTGTTCAATAGTATATACTCTGTATTTGAAAAATAGATGT60                ATATATTCTAGGTGATAAATTAAAAATGAAAGAATTTAATCATTGGAAAGTATTAAATAT120               ATATTGCTTATCTTCTCCAAGGAAGAGGAGTTCTCTCGTACCCATCCAAACTGACCTAAT180               TCTCAAGCTGCTTCATCTTGCTTGTACTGTAGGTTCATTTGCAATTTGTAGATTATGCTC240               CTTCAGGATTGGCTTTTGTAAATTTCTGTTAGAAGCTGGTTTCTGCATTTTTGATTTTTG300               TGTATTTGGATACATTTTCATATTGTGCAGAGAAATCCATGAGTTAAAAAATTATTTTTC360               CCTGTTTTATTTCTGCATGAACCTAAGTCACATTGACCCAGTAATTGATATATGTGTGAT420               TATTGCAATTAAGTATAAGAAGGTAGAATATATAGTTTTATTAGACAGATGCTTCCTGAA480               ATATTATTTTGTATGTTTTTACTATATCCTTTTTGTGTATCTACAGATACAACAGACATG540               CAAGAGAATGGACTCAGAAATATGCAATGTAAAAATCAAAAACATTTTCATATATAACCA600               GAGTACTGTAAAATCTAGGTTTTTTTTCAACATTAGCAGTAAATTGAGCACTGTTTACCT660               GTTTCATTGTACCATGAAACCATTTGATTTTTACCATTTTAAATGTGTCTCAAGCAAGAC720               AAAACAAACTTCCAAAAATACCCTTAAGACTGTGATGAGAGCATTTATCATTTTGTATGC780               ATTGAGAAAGACATTTATTATGGTTTTTAAGATACTTGGACATCTGCATCTTCAGCTTAC840               AAGATCTACAATGCAGCTGAAAAAGCAACCAAATTATTTTTTGCTGAAAACTAGATGTTT900               TTTACATGAGAAAATACTGTATGTGTGTCTAAGATGTCAGTTTTATAAATCTGTATTCAG960               ATTCATCCTTTTGTTAGCTCACTTTATAATTTGTATTTTTTTTCTGTATAGAACTAAATA1020              ACATGTATGTCAACTCATTACTTTTTTCCTGTGAACAGTATTGAAAACCCCAACCGGCTG1080              ATAATTAAGTGAATTAACTGTGTCTCCCTTGTCTTAGGATATTCTGTAGATTGATTGCAG1140              ATTTCTTAAATCTGAAATGACTTTACACTGTAATTCTCAGCATACTGATTATGGAGAACA1200              CTTGTTTTGAATTTTGTTATACTTGACTTAACTTTATTGCAATGTGAATTAATTGACTGC1260              TAAGTAGGAAGATGTGTAACTTTTATTTGTTGCTATTCACATTTGAATTTTTTCCTGTAT1320              AGGCAATATTATATTGACACCTTTTACAGATCTTACTGTAGCAAAAACCATATAAATAAA1380              ATGCTTTTTCTGCTA1395                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 337 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysGluPheAsnHisTrpLysValLeuAsnIleTyrCysLeuSer                              151015                                                                        SerProArgLysArgSerSerLeuValProIleGlnThrAspLeuIle                              202530                                                                        LeuLysLeuLeuHisLeuAlaCysThrValGlySerPheAlaIleCys                              354045                                                                        ArgLeuCysSerPheArgIleGlyPheCysLysPheLeuLeuGluAla                              505560                                                                        GlyPheCysIlePheAspPheCysValPheGlyTyrIlePheIleLeu                              65707580                                                                      CysArgGluIleHisGluLeuLysAsnTyrPheSerLeuPheTyrPhe                              859095                                                                        CysMetAsnLeuSerHisIleAspProValIleAspIleCysValIle                              100105110                                                                     IleAlaIleLysTyrLysLysValGluTyrIleValLeuLeuAspArg                              115120125                                                                     CysPheLeuLysTyrTyrPheValCysPheTyrTyrIleLeuPheVal                              130135140                                                                     TyrLeuGlnIleGlnGlnThrCysLysArgMetAspSerGluIleCys                              145150155160                                                                  AsnValLysIleLysAsnIlePheIleTyrAsnGlnSerThrValLys                              165170175                                                                     SerArgPhePhePheAsnIleSerSerLysLeuSerThrValTyrLeu                              180185190                                                                     PheGlyCysThrMetLysProPheAspPheTyrHisPheLysCysVal                              195200205                                                                     SerSerLysThrLysGlnThrSerLysAsnThrLeuLysThrValMet                              210215220                                                                     ArgAlaPheIleIleLeuTyrAlaLeuArgLysThrPheIleMetVal                              225230235240                                                                  PheLysIleLeuGlyHisLeuHisLeuGlnLeuThrArgSerThrMet                              245250255                                                                     GlnLeuLysLysGlnProAsnTyrPheLeuLeuLysThrArgCysPhe                              260265270                                                                     LeuHisGluLysIleLeuTyrValCysLeuArgCysGlnPheTyrLys                              275280285                                                                     SerValPheArgPheHisProLeuLeuAlaHisPheIleIleCysIle                              290295300                                                                     PhePheLeuTyrArgThrLysTyrIleLeuPheThrCysMetSerThr                              305310315320                                                                  HisTyrPhePheProValSerIleGluAsnProAsnArgLeuIleIle                              325330335                                                                     Gly                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated nucleotide sequence encoding the stimulator of Fe (iron) transport (SFT) protein.
 2. A method of producing the stimulator of Fe (iron) (SFT) transport protein comprising the steps of:a. inserting the nucleotide sequence of claim 1 into an expression vector; b. transforming in vitro host cells with the vector; c. expressing the protein in the host cells; and d. isolating the protein.
 3. An expression vector comprising the nucleotide sequence of claim
 1. 4. An in vitro host cell transformed by the expression vector of claim
 3. 5. An isolated nucleotide consisting of the nucleotide sequence of SEQ ID NO:
 1. 6. A method of production the stimulator of Fe (iron) (SFT) transport protein comprising the steps of:a. inserting the nucleotide sequence of claim 5 into an expression vector; b. transforming in vitro host cells with the vector; c. expressing the protein in the host cells; and d. isolating the protein.
 7. An expression vector comprising the nucleotide sequence of claim
 5. 8. An in vitro host cell transformed by the expression vector of claim
 7. 9. The host cell of claims 4 or 8 wherein the cell is a prokaryotic or eukaryotic cell. 